Non-specific Antibody Binding via Fc Receptors

Symptom

Elevated background staining on myeloid cells (monocytes, macrophages, dendritic cells, granulocytes) in bone marrow, blood, spleen, or in vitro myeloid cultures. False-positive signals not blocked by standard washing.

1. 1 Fcγ receptors on myeloid cells bind to antibody Fc regions independently of antigen specificity
2. 2 Bone marrow, blood, and spleen samples enriched in Fc receptor-expressing cells
3. 3 In vitro differentiated myeloid cultures express high levels of scavenger receptors
4. 4 Tandem dyes (PE/Dazzle 594, APC/Fire 750, PE/Cy7, PE/Cy5, PerCP/Cy5.5, especially APC/Cy7) bind monocytes/macrophages independent of Fc receptors

1. 1 Include FcR blocker (Fc receptor blocking reagent) in staining protocol when working with myeloid-enriched samples
2. 2 Add True-Stain Monocyte Blocker to minimize tandem dye binding to monocytes and macrophages
3. 3 Pre-incubate cells with blocking reagent for 10-15 minutes before antibody staining
4. 4 Use F(ab')2 fragments or Fab fragments lacking Fc region for critical markers
5. 5 Avoid assigning critical dim markers to APC/Cy7 when analyzing monocyte/macrophage-rich samples

Related Video (2)

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